Phosphatidylcholine transfer protein (PC-TP, a.k.a. StarD2) is a highly specific intracellular lipid binding protein with accentuated expression in liver and other oxidative tissues, including brown fat, heart, and skeletal muscle. Using PC-TP-deficient (Pctp-/-) mice, we have demonstrated that PC-TP regulates biliary lipid secretion, hepatic cholesterol homeostasis and high density lipoprotein (HDL) metabolism. In addition, mice lacking PC-TP expression are relatively resistant to developing atherosclerosis. In contrast to our original prediction that PC-TP transports phosphatidylcholines to the plasma membrane for export from hepatocytes, this research has suggested a more global regulatory role in hepatic lipid homeostasis. Preliminary studies in Pctp-/- mice have also revealed profound insulin-mediated decreases in hepatic glucose production, as well as altered energy substrate utilization by extrahepatic oxidative tissues. PC-TP may regulate lipid and glucose metabolism via direct interactions with the mitochondrial-associated protein thioesterase superfamily member 2 (Them2), potentially by controlling access of fatty acids to mitochondria. Specific Aim 1 will test the hypothesis that PC-TP regulates hepatic insulin sensitivity. We will examine whether lack of PC-TP expression protects against diet-induced insulin resistance and whether liver-specific expression of PC-TP reduces hepatic insulin sensitivity. Insulin signaling will be studied using cell culture systems in which PC-TP expression levels are broadly varied. We will also explore evidence both in vivo and in cell culture that PC-TP-Them2 interactions contribute to the control of insulin action. Specific Aim 2 will explore the influence of PC-TP on the differentiation and function of brown fat, which expresses both PC-TP and Them2. Gene expression, as well as cellular function and morphology will be characterized during the differentiation of brown pre-adipocytes cultured from Pctp-/- and wild type mice. Mechanisms by which PC-TP influences the maturation of brown adipocytes will be gleaned by systematically reintroducing PC-TP with or without Them2 during the process of differentiation. These data will be correlated with measurements of brown fat thermogenesis in vivo. Studies in cell culture will explore mechanisms by which PC-TP and Them2 activate key transcription factors in brown fat. Specific Aim 3 will investigate a mechanistic relationship between the in vitro and in vivo activities of PC-TP. High-throughput screening has identified small molecule inhibitors of the phosphatidylcholine transfer activity of PC-TP. This Specific Aim will continue the development of the most promising inhibitors. Mechanisms of inhibition will be elucidated and compounds will be tested in cells. Comparisons will be made to cells in which PC-TP expression is absent or knocked down. These studies should provide new insights into the regulation of lipid and glucose metabolism and may lead to a novel approach to managing insulin resistance, which constitutes the hallmark of the metabolic syndrome and non-alcoholic fatty liver disease. PUBLIC HEALTH RELEVANCE: Resistance to insulin is a hallmark of the metabolic syndrome and type II diabetes, which predispose to non-alcoholic fatty liver disease. The proposed studies will examine a key role for phosphatidylcholine transfer protein/StarD2, a highly specific lipid binding protein, in insulin- mediated regulation of lipid and glucose metabolism within the liver and energy consuming tissues of the body. It is anticipated that these experiments may establish phosphatidylcholine transfer protein/StarD2 as a molecular target for the management of insulin resistance.